The continuing goal of this grant is to understand at a molecular level the functions of the Ran GTPase, and of Ran-interacting proteins, in nuclear import and export. Substantial progress was made within the previous funding period to determine how the RanGTP gradient across the nuclear envelope is formed, to identify a new class of transport cofactors, and discover novel transport carriers. The following specific aims will build on these advances: 1. A combination of computational biology and experimental tests will be used to evaluate different protein import mechanisms, and to determine the rate-limiting steps in import, using intact cells under physiologically relevant conditions. 2. The roles of exportin-5 and of ILF3/NFAR in dsRNA binding and export will be elucidated. 3. The hypothesis will be tested that the transport cofactors RanBP3 and Npap60 function in cargo selection and nuclear pore association to regulate protein export and import. Nucleo-cytoplasmic transport is central to the function of the eukaryotic cell, and is a key regulatory step in signal transduction pathways that modulate gene expression and the cell cycle. Nuclear import and export are also critical to the life cycles of many pathogenic viruses, and nuclear transport pathways are frequently subverted by these agents to ensure preferential replication of the viral genome, and to inactivate anti-viral defense mechanisms. Understanding the mechanisms of nuclear transport at a molecular level may therefore provide important new targets for anti-viral therapies. [unreadable] [unreadable]